Method of efficiently detecting double-stranded nucleic acid

ABSTRACT

This invention relates to a method for efficiently detecting double-stranded nucleic acids. More particularly, this invention relates to a method for reducing signals derived from an intercalator bound to a single-stranded nucleic acid, wherein a compound that reacts more preferentially with an intercalator bound to a single-stranded nucleic acid than with an intercalator bound to a double-stranded nucleic acid or a compound that is bound to a single-stranded nucleic acid more strongly than an intercalator and is bound to a double-stranded nucleic acid more weakly than an intercalator is added to a mixture comprising double-stranded and single-stranded nucleic acids both having intercalators bound thereto, thereby reducing signals derived from an intercalator bound to a single-stranded nucleic acid.

TECHNICAL FIELD

The present invention relates to a method for detecting nucleic acidsthat can detect double-stranded nucleic acids using an intercalator withhigher sensitivity.

BACKGROUND ART

The most general method for detecting the amplification product obtainedby nucleic acid amplification such as polymerase chain reactions (PCR)is carried out by subjecting the solution after amplification to agarosegel electrophoresis and binding a fluorescent intercalator such asethidium bromide thereto, and then observing specific fluorescence. Whenthere is no possibility of contamination by other DNA and only theoccurrence of the amplification product is of interest, fluorescence canbe observed by adding a fluorescent intercalator to the solution afteramplification while omitting electrophoresis. A fluorescentintercalator, however, binds to a single-stranded DNA such as a primerand emits fluorescence. Accordingly, a significant level of backgroundnoise can be contained in the detected fluorescent signal.

Recently, the present inventors have succeeded in developing a novelmethod for nucleic acid amplification, which does not require thecomplicated temperature control that is supposedly inevitable in PCR,i.e., the loop-mediated isothermal amplification (LAMP) method (Notomi,T. et al., Nucleic Acids Res. 28 (12), e63 (2000), WO 00/28082). In theLAMP method, the 3′ terminal region of template polynucleotide isself-annealed, synthesis of complementary strands is started therefrom,and a primer that is annealed to the loop formed in the aforementionedsynthesis is used in combination therewith. This enables theamplification under isothermal conditions, and has remarkably enhancedthe simplicity of nucleic acid amplification.

In real-time monitoring of the product of nucleic acid amplificationusing a fluorescent intercalator, fluorescence intensity significantlyvaries in PCR since the product of nucleic acid amplification isrepeatedly dissociated and reassociated due to thermal denaturation as athermal cycle proceeds. In the LAMP method, however, fluorescenceintensity does not vary since the reaction proceeds under isothermalconditions. Thus, the LAMP method is more suitable for real-timemonitoring of the product of nucleic acid amplification. The LAMPmethod, however, requires approximately 10 times as many primers as thequantity required in PCR. When the product of nucleic acid amplificationobtained by the LAMP method is intended to be detected using afluorescent intercalator, the level of background noise caused bysingle-stranded primers, which are also present therein, is high. Thus,it is difficult to detect only the amplified double-stranded nucleicacids with high sensitivity.

An object of the present invention is to provide a process for detectingnucleic acids that can detect double-stranded nucleic acids using anintercalator with higher sensitivity by reducing signals derived from anintercalator bound to single-stranded nucleic acids.

The present inventors have conducted concentrated studies in order toattain the above object. As a result, they have succeeded in reducingsignals derived from an intercalator bound to a single-stranded nucleicacid with the addition of a compound that reacts more preferentiallywith an intercalator bound to a single-stranded nucleic acid than withan intercalator bound to a double-stranded nucleic acid or a compoundthat is bound to a single-stranded nucleic acid more strongly than anintercalator and is bound to a double-stranded nucleic acid more weaklythan an intercalator to a mixture comprising double-stranded andsingle-stranded nucleic acids both having intercalators bound thereto.This has led to the completion of the present invention.

More specifically, the present invention relates to a method forreducing signals derived from an intercalator bound to a single-strandednucleic acid, wherein a compound that reacts more preferentially with anintercalator bound to a single-stranded nucleic acid than with anintercalator bound to a double-stranded nucleic acid is added to amixture comprising double-stranded and single-stranded nucleic acidsboth having intercalators (e.g., ethidium bromide, acridine orange,TO-PRO-1® (Quinolinium,4-[(3-methyl-2(3H)-benzothiazolylidene)methyl]-1-[3-(trimethylammonio)propyl]-,diiodide, or YO-PRO-1® (Quinolinium,4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1-[3-(trimethylammonio)propyl]-,diiodide) bound thereto, thereby reducing signals derived from anintercalator bound to a single-stranded nucleic acid. Examples of acompound that reacts more preferentially with an intercalator bound to asingle-stranded nucleic acid than with an intercalator bound to adouble-stranded nucleic acid include an oxidant, such as sodiumhypochlorite, hydrogen peroxide, or potassium permanganate, and areducer, such as sodium borohydride or sodium cyanoborohydride.

Further, the present invention relates to a method for reducing signalsderived from an intercalator bound to a single-stranded nucleic acid,wherein a compound that is bound to a single-stranded nucleic acid morestrongly than an intercalator and is bound to a double-stranded nucleicacid more weakly than an intercalator is added to a mixture comprisingdouble-stranded and single-stranded nucleic acids both havingintercalators (e.g., ethidium bromide, acridine orange, TO-PRO-1®(Quinolinium,4-[(3-methyl-2(3H)-benzothiazolylidene)methyl]-1-[3-(trimethylammonio)propyl]-,diiodide), or YO-PRO-1® (Quinolinium,4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1-[3-(trimethylammonio)propyl]-,diiodide) bound thereto, thereby reducing signals derived from anintercalator bound to a single-stranded nucleic acid. An example of acompound that is bound to a single-stranded nucleic acid more stronglythan an intercalator and is bound to a double-stranded nucleic acid moreweakly than an intercalator is a second intercalator (e.g. methyleneblue, actinomycin D, SYBR® Green 2 (CAS Registry No. 172827-25-7), orOliGreen® (CAS Registry No. 268220-33-3)) different from the aboveintercalator.

Furthermore, the present invention relates to a method for detecting aproduct of nucleic acid amplification comprising the following-steps:

(a) amplifying a nucleic acid through nucleic acid amplification;

(b) adding an intercalator to a reaction solution after the nucleic acidamplification;

(c) reducing signals derived from an intercalator bound to asingle-stranded nucleic acid by any of the aforementioned methods; and

(d) assaying the fluorescence intensity of a reaction solution.

Further, the present invention relates to a method for detecting aproduct of nucleic acid amplification comprising the following steps:

(a) amplifying a nucleic acid through nucleic acid amplification in thepresence of an intercalator;

(b) reducing signals derived from an intercalator bound to asingle-stranded nucleic acid by any of the aforementioned methods; and

(c) assaying the fluorescence intensity of a reaction solution.

The present invention further relates to a method for detecting aproduct of nucleic acid amplification comprising the following steps:

(a) amplifying a nucleic acid through nucleic acid amplification in thepresence of an intercalator and a compound that is bound to asingle-stranded nucleic acid more strongly than an intercalator and isbound to a double-stranded nucleic acid more weakly than anintercalator; and

(b) assaying the fluorescence intensity of a reaction solution.

The nucleic acid amplification can be carried out by the followingsteps:

(a) selecting a first arbitrary sequence F1c, a second arbitrarysequence F2c, and a third arbitrary sequence F3c in that order from the3′ terminus in a target region toward the 3′ terminus on thepolynucleotide chain and a fourth arbitrary sequence R1, a fiftharbitrary sequence R2, and a sixth arbitrary sequence R3 in that orderfrom the 5′ terminus in the target region toward the 5′ terminus of thenucleotide chain;

(b) preparing a primer containing sequence F2 which is complementary toF2c and, on the 5′ side of F2, the same sequence as F1c; a primercontaining sequence F3 which is complementary to F3c; a primercontaining the same sequence as R2 and, on the 5′ side of the sequence,sequence R1c which is complementary to R1; and a primer containing thesame sequence as R3; and

(c) synthesizing DNA in the presence of a strand displacement-typepolymerase and the primers using the nucleotide chain as a template.

The nucleic acid amplification can be carried out by the followingsteps:

(a) selecting a first arbitrary sequence F1c and a second arbitrarysequence F2c in that order from the 3′ terminus in a target regiontoward the 3′ terminus on the polynucleotide chain and a third arbitrarysequence R1 and a fourth arbitrary sequence R2 in that order from the 5′terminus in the target region toward the 5′ terminus of the nucleotidechain;

(b) preparing a primer containing sequence F2 which is complementary toF2c and, on the 5′ side of F2, the same sequence as F1c; and a primercontaining the same sequence as R2 and, on the 5′ side of the sequence,sequence R1c which is complementary to R1; and

(c) synthesizing DNA in the presence of a strand displacement-typepolymerase, the primers, and a melting temperature regulator (such asbetaine or trimethylamine N-oxide) using the nucleotide chain as atemplate for amplification.

The present invention further relates to a kit for detectingdouble-stranded nucleic acids comprising, as elements, an intercalator(e.g, ethidiun, bromide, acridine orange, TO-PRO-1® (Quinolinium,4-[(3-methyl-2(3H)-benzothiazolylidene)methyl]-1-[3-(trimethylammonio)propyl]-,diiodide), or YO-PRO-1® (Quinolinium,4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1-[3-(trimethylammonio)propyl]-,diiodide) and a compound that reacts more preferentially with anintercalator bound to a single-stranded nucleic acid than with anintercalator bound to a double-stranded nucleic acid and/or a compoundthat is bound to a single-stranded nucleic acid more strongly than anintercalator and is bound to a double-stranded nucleic acid more weaklythan an intercalator. Examples of the compound that reacts morepreferentially with an intercalator bound to a single-stranded nucleicacid than with an intercalator bound to a double-stranded nucleic acidinclude an oxidant such as sodium hypochlorite, hydrogen peroxide, orpotassium permanganate, and a reducer, such as sodium borohydride orsodium cyanoborohydride. An example of the compound that is bound to asingle-stranded nucleic acid more strongly than an intercalator and isbound to a double-stranded nucleic acid more weakly than an intercalatoris a second intercalator (e.g. methylene blue, actinomycin D, SYBR®Green 2 (CAS Registry No. 172827-25-7), or OliGreen® (CAS Registry No.268220-33-3)) different from the aforementioned intercalator.

DISCLOSURE OF THE INVENTION

The present invention is hereafter described in detail.

The present invention relates to a method for detecting double-strandednucleic acids by detecting, with higher sensitivity, signals derivedfrom an intercalator bound to a double-stranded nucleic acids throughthe reduction of signals derived from an intercalator bound to asingle-stranded nucleic acid with the addition of a compound that reactsmore preferentially with an intercalator bound to a single-strandednucleic acid than with an intercalator bound to a double-strandednucleic acid or a compound that is bound to a single-stranded nucleicacid more strongly than an intercalator and is bound to adouble-stranded nucleic acid more weakly than an intercalator to amixture comprising double-stranded and single-stranded nucleic acidsboth having intercalators bound thereto.

Accordingly, this method is particularly useful when selectivelydetecting amplification products when significant amounts of primersbesides the amplified double-stranded nucleic acids remain in thereaction system during or after the nucleic acid amplification thatutilizes primers as single-stranded nucleic acids.

In the present invention, the term “intercalator” refers to anintercalating agent, which is a compound that can be inserted(intercalated) in between adjacent planes formed by DNA nucleotidepairing. The term “signal” refers to a substance that functions as amarker for a specific substance or condition, such as fluorescencederived from an intercalator bound to a nucleic acid.

1. Nucleic Acid Amplification

Specifically, the present invention is useful when detecting anamplification product (double-stranded nucleic acid) obtained in thenucleic acid amplification that utilizes a primer, which is asingle-stranded nucleic acid. Examples of nucleic acid amplificationinclude, in addition to polymerase chain reaction (PCR), the LAMPmethod, the strand displacement amplification (SDA) method (JP PatentPublication (Kokoku) No. 7-114718 B (1995)), and the nucleic acidsequence based amplification (NASBA) method (JP Patent No. 2650159).More particularly, since a larger amount of primers are used in the LAMPmethod than in PCR, the amount of single-stranded primers remainingafter the amplification is large. Accordingly, the method for detectingdouble-stranded nucleic acids according to the present invention is veryuseful when detecting amplification products obtained by the LAMPmethod.

In the LAMP method, a loop structure is formed at a terminus of thenucleotide sequence to be amplified and, simultaneously with elongationby polymerase starting therefrom, a primer hybridized in a region withinthe loop dissolves the elongation product into a single strand whileelongating a nucleic acid chain by strand displacement. Since thegenerated single-stranded nucleic acid has a self-complementary regionat its terminus, it forms a loop at the terminus, and new elongation isinitiated. The actual LAMP method proceeds under isothermal conditionsand, thus, the reactions described above occur simultaneously and inparallel. The LAMP method is characterized by a very large amount of theamplification product in addition to a strand displacement-type reactionthat proceeds under isothermal conditions. One of the reasons for thisis that the LAMP method does not involve thermal denaturation, whichdeactivates polymerase. The LAMP method is hereafter described.

(1) LAMP Method

At the outset, a scheme of the LAMP method is shown (FIG. 1 and FIG. 2).In the LAMP method, a template polynucleotide, which is the target ofamplification, is prepared. The template polynucleotide (DNA or RNA) canbe prepared by chemical synthesis, or, in accordance with conventionalmethods, from biological materials such as tissues or cells. Thetemplate polynucleotide is prepared so that suitable lengths ofsequences (referred to as “bilateral sequences”) are present on thesides (5′ side and 3′ side) in the target region for amplification (FIG.1A). The term “bilateral sequence” refers to a sequence comprising aregion from the 5′ terminus in the target region to the 5′ terminus ofthe polynucleotide chain and a sequence comprising a region from the 3′terminus in the target region to the 3′ terminus of the polynucleotidechain (a portion indicated by two-headed arrows (← →) in FIG. 1A). Thelengths of the bilateral sequences are 10 to 1,000 nucleotides, andpreferably 30 to 500 nucleotides on the 5′ side and the 3′ side in thetarget region.

Predetermined regions are arbitrarily selected from the bilateralsequences in the template polynucleotide chain (FIG. 1A) containing thetarget region and the bilateral sequences. Specifically, a firstarbitrary sequence F1c, a second arbitrary sequence F2c, and a thirdarbitrary sequence F3c are selected in that order from the 3′ terminusin the target region toward the 3′ terminus of the polynucleotide chain(FIG. 1B). Similarly, a fourth arbitrary sequence R1, a fifth arbitrarysequence R2, and a sixth arbitrary sequence R3 are selected in thatorder from the 5′ terminus in the target region toward the 5′ terminusof the polynucleotide chain (FIG. 1B). When selecting the arbitrarysequence F1c and the arbitrary sequence R1, the distance between F1c andR1 can be 0 nucleotides, i.e., contiguous. Alternatively, it can beselected in such a manner that F1c and R1 are allowed to partiallyoverlap. The first to the sixth regions are respectively and arbitrarilyselected in accordance with the sequences of prepared polynucleotidechains. Each region to be selected comprises preferably 5 to 100nucleotides, and more preferably 10 to 50 nucleotides. Selection of thenucleotide length facilitates annealing of the primer described below.

Each of the arbitrary sequences is preferably selected so that, insteadof intermolecular annealing, the amplification product obtained by theLAMP method preferentially initiates the intramolecular annealingbetween sequence F1c and sequence F1 and between sequence R1 andsequence R1c as shown in FIG. 2L, and forms a terminal loop structure.For example, in order to preferentially initiate the intramolecularannealing, it is important to consider the distance between sequence F1cand sequence F2c and the distance between sequence R1 and sequence R1cwhen selecting the arbitrary sequences. More specifically, bothsequences are preferably located within a distance of 0 to 500nucleotides, preferably 0 to 100 nucleotides, and most preferably 10 to70 nucleotides. Numerical values respectively represent the number ofnucleotides without containing sequences F1c and F2c and sequences R1and R2.

Subsequently, a primer referred to as the “FA primer” is designed andsynthesized, and this is annealed to F2c. The term “FA primer” includessequence F2 which is complementary to region F2c and another sequencewhich is the same as F1c (this may be referred to as “F1c” forconvenience). Examples thereof include those having a structure in whichthe 3′-terminus of sequence F1c is linked to the 5′ side of F2 (FIG.1C). The term “annealing” refers to the formation of a double-strandstructure of a nucleotide chain through nucleotide pairing based on theWatson-Crick model. After the FA primer is annealed to sequence F2c onthe template polynucleotide chain, DNA strand synthesis is initiatedstarting from F2 in the FA primer (FIG. 1D). Subsequently, a primercontaining sequence F3, which is complementary to F3c (hereafter thismay be referred to as “F3 primer”), is annealed to sequence F3c on thetemplate polynucleotide chain (FIG. 1D). Strand displacement-typesynthesis of DNA is then carried out starting from the annealed F3primer (FIG. 1E). When a double-strand structure, which has beenproduced through the hybridization of a polynucleotide to a template forthe synthesis of a complementary chain, is subjected to a reaction thatsynthesizes, starting from a primer, a complementary chain whileseparating the polynucleotide from the template, this process is termed“strand displacement-type synthesis of DNA.” Specific examples thereofinclude a reaction in which synthesis proceeds so as to displace thechain synthesized by the FA primer with the chain synthesized by the F3primer. In other words, the complementary chain of the templatepolynucleotide chain synthesized by the FA primer can be displaced by achain elongated from the F3 primer in such a manner that thecomplementary chain is separated.

Two types of nucleotide chains, the following (i) and (ii), can beobtained by the above-described synthesis.

(i) A nucleotide chain containing sequence “(5′)F3-F2-F1-targetregion-R1c-R2c-R3c(3′),” which is complementary to sequence“(3′)F3c-F2c-F1c-target region-R1-R2-R3(5′)” in the templatepolynucleotide chain (FIG. 1F).

(ii) A nucleotide chain formed into a single strand by displacement(separated), i.e., a nucleotide chain containing “(5′)F1c-F2-F1-targetregion-R1c-R2c-R3c(3′)” having the same sequence as F1c on its 5′terminal side (FIG. 1G).

F1 and F1c are complementary to each other in the nucleotide chainaccording to (ii) above and, thus, they hybridize to each other based onthe intrachain hydrogen bonds between F1 and F1c, thereby forming ahairpin loop (FIG. 1G). F2 is contained in this hairpin loop.

Subsequently, a primer referred to as the “RA primer” is annealed tosequence R2c in the nucleotide chain according to (ii) above. In the RAprimer, the 3′ side of sequence R1c complementary to sequence R1 islinked to the 5′ side of sequence R2. DNA strand synthesis is theninitiated starting from the RA primer (FIG. 1H). When the elongated DNAsynthesized starting from the RA primer has reached the end of thedouble-strand chain formed between F1 and F1c, the sequence of F1c isdisplaced with the elongated DNA in the same manner as the displacementshown in FIG. 1E (FIG. 1I). A primer containing sequence R3, which iscomplementary to sequence R3c (hereafter it may be referred to as the“R3 primer”), is then annealed to R3c of the template polynucleotidechain (FIG. 1I). Strand displacement-type synthesis of DNA is thencarried out starting from the annealed R3 primer (FIG. 2J). Two types ofnucleotide chains, i.e., the following (iii) and (iv), are synthesizedbased on the above synthesis.

(iii) A nucleotide chain “(3′)F1-F2c-F1c-target region-R1-R2-R3(5′),”which is complementary to sequence “(5′)F1c-F2-F1-targetregion-R1c-R2c-R3c(3′)” (FIG. 2K).

(iv) A nucleotide chain “(3′)F1-F2c-F1-target region-R1-R2-R1c(3′)”having F1 located closest to the 3′ terminal side, and R1c locatedclosest to the 5′ terminal side (FIG. 2L).

The sequence according to (iv) above forms a hairpin loop by theintrachain hydrogen bonds between sequences F1 and F1c existing on the3′ side and between sequences R1 and R1c on the 5′ side (FIG. 2L).

Subsequently, among the nucleotide chains according to (iv) above,region F2 of the FA primer is annealed to F2c in the hairpin loopportion on the 3′ side (FIG. 2M). DNA strand synthesis is initiatedstarting from F1 annealed by the intrachain hydrogen bonds. In FIG. 1M,the elongation chain synthesized starting from F1 reaches the 5′terminus by opening the hairpin loop formed by R1-R2-R1c. In contrast,when a reaction proceeds starting from F2, a chain, which iscomplementary to a chain constituted by “F1c-target region-R1-R2-R1c,”is synthesized. In this case, F1 and the chain “F1-targetregion-R1c-R2c-R1” synthesized starting from F1 are displaced by thechain that is synthesized starting from F2. This provides fordouble-stranded DNA having a single-strand protrusive constructiondenoted as “-target sequence-R1c-R2c-R1.” The portion having asingle-strand protrusive construction forms a hairpin loop by formingintrachain hydrogen bonds between R1c and R1 of a portion having asingle-strand protrusive construction (“R1c-R2c-R1”) (FIG. 2N). Thisconstruct initiates DNA strand synthesis starting from R1 annealed bythe intrachain hydrogen bonds (FIG. 2N). Two types of nucleotide chains,the following (v) and (vi), are obtained based on the above synthesis.

(v) Sequence “(3′)R1-R2-R1 c-target region-F1-F2-F1c-targetregion-R1-R2c-R1c-target region-F1-F2c-F1c-target region-R1-R2-R1c(5′)”(FIG. 2O).

(vi) A sequence having F1c located closest to the 3′ terminal side andR1 located closest to the 5′ terminal side “(3′)F1c-F2-F1-targetregion-R1c-R2c-R1(3′)” (FIG. 2P).

The nucleotide chains according to (v) and (vi) above respectively forma hairpin loop having R2c as a loop portion and a hairpin loop having F2and R2c as another loop portion by intrachain hydrogen bonds. The RAprimer is annealed to the portion R2c forming the hairpin loop in twosequences, i.e., (v) and (vi) above, synthesis of DNA starting from theprimer is initiated, and synthesis of nucleotides chain (complementarychain with sequence shown in (vi)) containing a target sequenceproceeds. This complementary chain is the same as the sequence shown inFIG. 2L and, thus, the reactions according to FIGS. 2L to 2P arethereafter repeated. In contrast, the reaction from FIG. 1A can proceedand, thus, amplification of polynucleotide chain proceeds by repeatingthis series of syntheses.

The above-described amplification is carried out using four types ofprimers, i.e., the FA primer, the RA primer, the F3 primer, and the R3primer. Alternatively, amplification under isothermal conditions can beinitiated by using only two types of primers, the FA primer and the RAprimer, without using the F3 primer and the R3 primer. In thisalternative amplification, a melting temperature (Tm) regulator, forexample, betaine or trimethylamine N-oxide (TMANO), should be present inthe reaction system.

(2) Reaction Condition

In the reaction in accordance with the LAMP method, the ingredientsbelow are added to a template single-stranded nucleic acid:

(i) four types of oligonucleotides (FA, RA, outer primer F3, and outerprimer R3);

(ii) DNA polymerase for strand displacement-type synthesis ofcomplementary chains; and

(iii) a nucleotide serving as a substrate for DNA polymerase.

The reaction proceeds through incubation at such a temperature thatstable nucleotide pairing between a nucleotide sequence constituting FAor RA and a complementary nucleotide sequence thereof can be formed, andenzyme activity can be maintained. The incubation temperature is 50 to75° C., and preferably 55 to 70° C. The incubation time is 1 minute to10 hours, and preferably 5 minutes to 4 hours.

In the LAMP method according to the above two embodiments, the FA primerand the RA primer are also referred to as “inner primers” and the F3primer and the R3 primer are also referred to as “outer primers.”

Synthesis of nucleotide chains from the outer primer should be initiatedafter synthesis of nucleotide chains from the inner primer. A method forsatisfying this condition includes the one which sets the concentrationof the inner primer higher than that of the outer primer. Morespecifically, the concentration of the inner primer can be set higherthan that of the outer primer by 2- to 50-fold, and preferably by 4- to25-fold.

Polymerase, which catalyzes the strand displacement-type synthesis ofcomplementary chains (this may be referred to as “stranddisplacement-type polymerase), includes Bst DNA polymerase, Bca(exo-)DNA polymerase, the Klenow fragment of E. coli DNA polymerase I, VentDNA polymerase, Vent(Exo-) DNA polymerase (exonuclease activity isremoved from Vent DNA polymerase), DeepVent DNA polymerase,DeepVent(Exo-) DNA polymerase (exonuclease activity is removed fromDeepVent DNA polymerase), φ29 phage DNA polymerase, MS-2 phage DNApolymerase, Z-Taq DNA polymerase (Takara Shuzo Co., Ltd.), and KOD DNApolymerase (Toyobo Co., Ltd.).

This reaction is conducted in the presence of, for example, a buffergiving suitable pH to the enzyme reaction, salts necessary formaintaining the catalytic activity of the enzyme or for annealing, aprotective agent for the enzyme, and, if necessary, a regulator formelting temperature (Tm). A buffer, such as Tris-HCl having a bufferingaction in the range of weakly alkaline to neutral, is used. The pH isadjusted depending on the DNA polymerase being used. As salts, MgCl₂,KCl, NaCl, (NH₄)₂SO₄, etc. are suitably added to maintain the activityof the enzyme and to regulate the melting temperature (Tm) of thenucleic acid. Bovine serum albumin or sugars can be used as protectiveagents for enzymes. Further, betaine (N,N,N-trimethylglycine),trimethylamine N-oxide (TMANO), dimethyl sulfoxide (DMSO), or formamideis used as a regulator for melting temperature (Tm). By the use of theregulator for melting temperature (Tm), annealing of the oligonucleotidecan be regulated under restricted temperature conditions. In particular,betaine and trimethylamine N-oxide (TMANO) are also effective forimproving the efficiency of strand displacement by virtue of itsisostabilization properties. By adding betaine in an amount of 0.2 to3.0 M, and preferably about 0.5 to 1.5 M to the reaction solution, itspromoting action on the nucleic acid amplification of the presentinvention can be expected. Because these regulators for meltingtemperature act to lower melting temperature, conditions giving suitablestringency and reactivity must be empirically determined inconsideration of other reaction conditions such as concentration ofsalts and reaction temperature.

2. Methods for Reducing Signals Derived from an Intercalator Bound to aSingle-stranded Nucleic Acid

The following methods are examples of methods for reducing signalsderived from an intercalator bound to a single-stranded nucleic acid ina mixture comprising double-stranded and single-stranded nucleic acidsboth having intercalators bound thereto.

(1) A Method in Which a Compound that Reacts More Preferentially with anIntercalator Bound to a Single-stranded Nucleic Acid than with anIntercalator Bound to a Double-stranded Nucleic Acid is Added

(1)-1 Utilization of an Oxidant or Reducer

A compound that reacts more preferentially with an intercalator bound toa single-stranded nucleic acid than with an intercalator bound to adouble-stranded nucleic acid is added to a mixture comprisingdouble-stranded and single-stranded nucleic acids having intercalatorsbound thereto. This can reduce signals derived from an intercalatorbound to a single-stranded nucleic acid. An example eta compound thatreacts more preferentially with an intercalator bound to asingle-stranded nucleic acid than with an intercalator bound to adouble-stranded nucleic acid is an oxidant or reducer. Specifically, anoxidant or reducer is further added to a mixed solution ofdouble-stranded and single-stranded nucleic acids that is stained by theaddition of an intercalator, and the resultant is maintained at suitabletemperature for a suitable period of time. Thus, the intercalator boundto single-stranded nucleic acids is preferentially oxidized or reducedcompared with the intercalator bound to double-stranded nucleic acids.This lowers fluorescence intensity derived from the intercalator boundto a single strand. Examples of an intercalator include ethidiumbromide, acridine orange, TO-PRO-1® (Quinolinium,4-[(3-methyl-2(3H)-benzothiazolylidene)methyl]-1-[3-(trimethylammonio)propyl]-,diiodide), and YO-PRO-1® (Quinolinium,4-[(3-methyl-2(3H)benzoxazolylidene)methyl]-1-[3-(trimethylammonio)propyl]-,diiodide. Examples of an oxidant include sodium hypochlorite. (NaClO),hydrogen peroxide (H₂O₂), and potassium permanganate (KMnO₄). Examplesof a reducer include sodium borohydride (NaBH₄) and sodiumcyanoborohydride (NaBH₃CN).

When detecting a product of nucleic acid amplification by this method,fluorescence intensity is generally assayed through addition of anintercalator to the reaction solution after the nucleic acidamplification, followed by further addition of an oxidant or reducer.Alternatively, an intercalator is added to the reaction solution beforethe nucleic acid amplification, amplification is carried out in thepresence of the intercalator, and an oxidant or reducer is added afterthe reaction, thereby assaying the fluorescence intensity.

(1)-2 Utilization of a Complex-forming Compound

A compound that forms a complex with an intercalator can be used as acompound that reacts more preferentially with an intercalator bound to asingle-stranded nucleic acid than with an intercalator bound to adouble-stranded nucleic acid. Specifically, an intercalator and thecomplex-forming compound are added to a mixed solution ofdouble-stranded and single-stranded nucleic acids, and the resultant ismaintained at suitable temperature for a suitable period of time. A weakbond between a single-stranded nucleic acid and an intercalator ispreferentially inhibited by the complex-forming reaction compared with abond between a double-stranded nucleic acid and an intercalator. Thislowers fluorescence intensity derived from an intercalator bound to asingle strand. An example of a combination of an intercalator and acomplex-forming compound is that of methylene blue (an intercalator) andAcid Orange 7 (a complex-forming compound).

When detecting a product of nucleic acid amplification by this method,fluorescence intensity is generally assayed through simultaneous orsequential addition of an intercalator and a complex-forming compound tothe reaction solution after the nucleic acid amplification.Alternatively, an intercalator is added to the reaction solution beforethe nucleic acid amplification, amplification is carried out in thepresence of the intercalator, and a complex-forming compound is addedafter the reaction, thereby assaying the fluorescence intensity.

Absorbance may be assayed instead of fluorescence intensity by utilizingdifferences in the absorption spectrum caused by the complex formation.That is, since the absorption spectrum of an intercalator differs fromthat of the complex thereof, it is possible to quantitatively assay onlythe intercalator that did not form a complex.

(2) A Method in Which a Compound that is Bound to a Single-strandedNucleic Acid More Strongly than an Intercalator and is Bound to aDouble-stranded Nucleic Acid More Weakly than an Intercalator is Added

A compound that is bound to a single-stranded nucleic acid more stronglythan an intercalator and is bound to a double-stranded nucleic acid moreweakly than an intercalator is added to a mixture comprisingdouble-stranded and single-stranded nucleic acids both havingintercalators bound thereto. This can reduce signals derived from anintercalator bound to a single-stranded nucleic acid. An example of acompound that is bound to a single-stranded nucleic acid more stronglythan an intercalator and is bound to a double-stranded nucleic acid moreweakly than an intercalator is a second intercalator that is differentfrom the aforementioned intercalator (hereafter referred to as a “firstintercalator”). A second intercalator different from the firstintercalator that is bound to a single-stranded nucleic acid morestrongly than the first intercalator and is bound to a double-strandednucleic acid more weakly than the first intercalator is added to a mixedsolution of double-stranded and single-stranded solution which isstained by the addition of the first intercalator, and the resultant ismaintained at suitable temperature for a suitable period of time. Thus,the first intercalator bound to a single-stranded nucleic acid ispreferentially displaced by the second intercalator compared with thefirst intercalator bound to a double-stranded nucleic acid. This lowersfluorescence intensity derived from the first intercalator bound to asingle strand. Examples of the first intercalator that is first added toa mixture of double-stranded and single-stranded nucleic acids includeethidium bromide, acridine orange, TO-PRO-1® (Quinolinium,4-[(3-methyl-2(3H)-benzothiazolylidene)methyl]-1-[3-(trimethylammonio)propyl]-,diiodide), and YO-PRO-1® (Quinolinium,4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1-[3-(trimethylammonio)propyl]-,diiodide. Examples of the second intercalator, which is added for thepreferential displacement with the first intercalator bound to asingle-stranded nucleic acid and is different from the firstintercalator, include methylene blue, actinomycin D, SYBR® Green 2 (CASRegistry No. 172827-25-7), and OliGreen® (CAS Registry No. 268220-33-3).

When detecting a product of nucleic acid amplification by this method,fluorescence intensity is generally assayed through addition of anintercalator to the reaction solution after the nucleic acidamplification, followed by further addition of the second intercalatordifferent from the aforementioned intercalator. Alternatively, anintercalator is added to the reaction solution before the nucleic acidamplification, amplification is carried out in the presence of theintercalator, and the second intercalator different from theaforementioned intercalator is added after the reaction, therebyassaying the fluorescence intensity. Further, the two above types ofintercalators are previously added to the reaction solution before thenucleic acid amplification, amplification is carried out in the presenceof the two types of intercalators, and fluorescence intensity can beassayed after the reaction.

3. Detection of Double-stranded Nucleic Acids

In the present invention, double-stranded nucleic acids are detected byassaying the fluorescence emitted from the intercalator bound to nucleicacids using a fluorophotometer after the process described in 2 above.For example, when detecting the amplification product obtained in thereaction in 1 above, a reaction system in which amplification wascarried out without template DNA (control system 1) or a reaction systemwithout DNA polymerase (control system 2) is provided as a control. Theaforementioned control system and a system in which amplification wascarried out as usual in the presence of template DNA and DNA polymerase(a test system) are subjected to the process as described in 2 above,and differences in fluorescence intensity between the control system andthe test system are inspected. Thus, the generation of the amplificationproduct in the reaction solution through the reaction can be confirmed.More specifically, when fluorescence intensity of the test system isgreater than that of the control system 1 or 2, it can be evaluated thatthe amplification product was detected in the test system.

An example of a fluorophotometer that can be used for assayingfluorescence intensity is the ABI PRISM 7700 sequence detection system(PE Applied Biosystems). When assaying fluorescence intensity, theexcitation wavelength and the assay wavelength are suitably determinedin accordance with the type of intercalator used for staining nucleicacids. In the present invention, the reaction solution after theinitiation of amplification is subjected to the process described in 2above, and fluorescence intensity thereof is then assayed with theelapse of time. Thus, transition in the conditions of the reactionproduct with the elapse of the reaction time can be monitored.

4. Kit for Detecting or Monitoring Double-stranded Nucleic Acids

In the method for detecting or monitoring the product of nucleic acidamplification according to 3 above, reagents necessary forimplementation can be packaged and supplied as a kit Specific examplesinclude a kit comprising the following elements.

[Elements of Kit]

(1) An intercalator (for example, ethidium bromide, acridine orange,TO-PRO-1® (Quinolinium,4-[(3-methyl-2(3H)-benzothiazolylidene)methyl]-1-[3-(trimethylammonio)propyl]-,diiodide), and YO-PRO-1® (Quinolinium,4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1-[3-(trimethylammonio)propyl]-,diiodide);

(2) a compound that reacts more preferentially with an intercalatorbound to a single-stranded nucleic acid than with an intercalator boundto a double-stranded nucleic acid (for example, an oxidant such assodium hypochlorite (NaClO), hydrogen peroxide (H₂O₂), or potassiumpermanganate (KMnO₄) and a reducer such as sodium borohydride); and

(3) a compound tat is bound to a single-stranded nucleic acid morestrongly than an intercalator and is bound to a double-stranded nucleicacid more weakly than an intercalator (for example, the secondintercalator different from the intercalator that was first added to amixed solution of double-stranded and single-stranded nucleic acids suchas methylene blue, actinomycin D, SYBR® Green 2 (CAS Registry No.172827-25-7), or OliGreen® (CAS Registry No. 268220-33-3)).

This kit can also be used for amplifying and detecting a target nucleicacid based on the LAMP method by adding the elements below:

[Elements that Can be Added]

(a) when a first arbitrary sequence F1c, a second arbitrary sequenceF2c, and a third arbitrary sequence F3c are selected in that order fromthe 3′ terminus in the target region toward the 3′ terminus of thepolynucleotide chain that constitutes a nucleic acid to be detected anda fourth arbitrary sequence R1, a fifth arbitrary sequence R2, and asixth arbitrary sequence R3 are selected in that order from the 5′terminus in the target region toward the 5′ terminus of thepolynucleotide chain, a primer containing sequence F2 which iscomplementary to F2c and, on the 5′ side of F2, the same sequence asF1c; a primer containing sequence F3 which is complementary to F3c; aprimer containing the same sequence as R2 and, on the 5′ side of thesequence, sequence R1c which is complementary to R1; and a primercontaining the same sequence as R3;

(b) a polymerase catalyzing strand displacement-type synthesis ofcomplementary chains; and

(c) a nucleotide serving as a substrate for the synthesis ofcomplementary strands.

The elements of the kit can vary according to the embodiment of the LAMPmethod to be employed. Specifically, a primer containing the sequence F3which is complementary to arbitrary sequence F3c and a primer containingthe same sequence as arbitrary sequence R3 can be optionally omittedfrom the elements. In such a case, a melting temperature regulator (forexample, betaine or trimethylamine N-oxide) is preferably added.Further, a buffer providing suitable conditions for the enzyme reactionand reagents necessary for detecting the reaction product of synthesismay be optionally added. According to a preferred embodiment of thepresent invention, reagents necessary for one reaction can be suppliedin the state of being fractionated into reaction vessels.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a scheme of amplification by the LAMP method.

FIG. 2 shows a scheme of amplification by the LAMP method.

FIG. 3 shows fluorescence intensity of each reaction solution when theLAMP reaction is carried out in the presence of ethidium bromide,followed by the addition of a reducer or oxidant.

FIG. 4 shows fluorescence intensity of each reaction solution when theLAMP reaction is carried out in the presence of acridine orange,followed by the addition of a reducer or oxidant.

FIG. 5 shows fluorescence intensity of each reaction solution when theLAMP reaction is carried out by adding the second intercalator inaddition to ethidium bromide.

FIG. 6 shows fluorescence intensity of each reaction solution when theLAMP reaction is carried out by adding the second intercalator inaddition to acridine orange.

FIG. 7 shows fluorescence intensity of each reaction solution when theLAMP reaction is carried out by adding methylene blue as the secondintercalator to YO-PRO-1® (Quinolinium,4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1-[3-(trimethylammonio)propyl]-,diiodide.

FIG. 8 shows absorbance of each reaction solution when the LAMP reactionis carried out by adding methylene blue and Acid Orange 7.

This description includes part or all of the contents as disclosed inthe description of Japanese Patent Application No. 2001-183716, which isa priority document of the present application.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be described in more detail with reference tothe following examples, although the technical scope of the presentinvention is not limited to these examples.

EXAMPLE 1 Effect of an Oxidant or Reducer On the Detection of the LampReaction Product Using Ethidium Bromide

(1) Nucleic Acid Amplification by the LAMP Method

TABLE 1 Composition of reaction solution Composition of reactionsolution (in 25 μL) 20 mM Tris-HCl pH 8.8 10 mM KCl 10 mM (NH₄)₂SO₄ 4 mMMgSO₄ 0.1% Tween 20 0.4 mM dNTP 8 U Bst DNA polymerase (NEB) 1.6 μM FAprimer 1.6 μM RA primer 0.4 μM F3 primer 0.4 μM R3 primer

To the above reaction solution, 6×10⁻²⁰ mol of DNA of prostate-specificantigen (PSA) as a template for the LAMP reaction and ethidium bromide(EtBr, 0.5 μ/ml) for detecting amplification products were added.Amplification was then carried out at 65° C. for 30 minutes. Thereaction solution to which template DNA had been added was determined tobe a positive reaction solution, and the reaction solution without theaddition of template DNA was determined to be a negative reactionsolution. In this amplification, the following sequence (SEQ ID NO: 1)included in the template was determined to be a polynucleotide ofinterest.

5′-TGCTTGTGGCCTCTCGTGGCAGGGCAGTCTGCG (SEQ ID NO: 1)GCGGTGTTCTGGTGCACCCCCAGTGGGTCCTCACAGCTGCCCACTGCATCAGGAACAAAAGCGTGATCTTGCTGGGTCGGCACAGCCTGTTTCATCCTGAAGACACAGGCCAGGTATTTCAGGTCAGCCACAGCTTCACACACC C-3′

Inner primers (FA primer and RA primer) and outer primers (F3 primer andR3 primer) in the reaction solution were designed as follows based onthe nucleotide sequence as shown in SEQ ID NO: 1.

[Inner primers] FA primer 5′-TGTTCCTGATGCAGTGGGCAGCTTTAGTCTGCG (SEQ IDNO: 2) GCGGTGTTCTG-3′ RA primer 5′-TGCTGGGTCGGCACAGCCTGAAGCTGACCTGAA(SEQ ID NO: 3) ATACCTGGCCTG-3′ [Outer primers] F3 primer5′-TGCTTGTGGCCTCTCGTG-3′ (SEQ ID NO: 4) R3 primer5′-GGGTGTGTGAAGCTGTG-3′ (SEQ ID NO: 5)(2) Oxidation or Reduction

The positive and the negative reaction solutions after the amplificationwere subjected to oxidation or reduction. Specifically, oxidation wascarried out by adding sodium hypochlorite (NaClO) to the both reactionsolutions to concentrations of 2.8% and maintaining the resultant atroom temperature for 2 hours. Reduction was carried out by adding 4 mMsodium borohydride (NaBH₄) and 0.4 mM sodium hydroxide (NaOH) to theboth reaction solutions and maintaining the resultant on ice for 10minutes. After the reaction, the fluorescence intensity of each reactionsolution was assayed using the ABI PRISM® 7700 sequence detection system(PE Applied Biosystems) at an excitation wavelength of 488 nm and anassay wavelength of 605 nm.

The assay results are shown in FIG. 3. In the case of the reactionsolutions which were not subjected to oxidation or reduction (EtBr inFIG. 3), the fluorescence intensity of the positive reaction solutionwas 3,012, and that of the negative reaction solution was 2,695. Thatis, difference in the fluorescence intensity resulting from theoccurrence of products of double-stranded nucleic acid amplification was317. In contrast, in the case of the reaction solutions which weresubjected to oxidation using sodium hypochlorite (EtBr+NaClO in FIG. 3),the fluorescence intensity of the positive reaction solution was 1,784,and that of the negative reaction solution was 648. That is, differencein the fluorescence intensity resulting from the occurrence of productsof double-stranded nucleic acid amplification was as large as 1,136. Inthe case of the reaction solutions which were subjected to reductionusing sodium borohydride (EtBr+NaBH₄ in FIG. 3), the fluorescenceintensity of the positive reaction solution was 1,051, and that of thenegative reaction solution was 218. That is, difference in thefluorescence intensity resulting from the occurrence of products ofdouble-stranded nucleic acid amplification was as large as 833.

Thus, sensitivity for detecting products of double-stranded nucleic acidamplification using ethidium bromide can be enhanced by oxidizing orreducing the amplification products stained with ethidium bromide.

EXAMPLE 2 Effect of an Oxidant or Reducer on the Detection of the LAMPReaction Product Using Acridine Orange

The effects of an oxidant or reducer on the efficiency of detecting theLAMP reaction product were inspected under the same experimentalconditions as used in Example 1 except that acridine orange was addedinstead of ethidium bromide and the assay wavelength was set at 575 nmin the amplification of nucleic acids described in Example 1. The assayresults are shown in FIG. 4. In the case of the reaction solutions whichwere not subjected to oxidation or reduction (AO in FIG. 4), thefluorescence intensity of the positive reaction solution was 7,053, andthat of the negative reaction solution was 6,155. That is, difference inthe fluorescence intensity resulting from the occurrence of products ofdouble-stranded nucleic acid amplification was 898. In contrast, in thecase of the reaction solutions which were subjected to oxidation usingsodium hypochlorite (AO+NaClO in FIG. 4), the fluorescence intensity ofthe positive reaction solution was 5,521, and that of the negativereaction solution was 201. That is, difference in the fluorescenceintensity resulting from the occurrence of products of double-strandednucleic acid amplification was as large as 5,320. In the case of thereaction solutions which were subjected to reduction using sodiumborohydride (AO+NaBH₄ in FIG. 4), the fluorescence intensity of thepositive reaction solution was 6,214, and that of the negative reactionsolution was 596. That is, difference in the fluorescence intensityresulting from the occurrence of products of double-stranded nucleicacid amplification was as large as 5,618.

Thus, sensitivity for detecting products of double-stranded nucleic acidamplification using acridine orange can be enhanced by oxidizing orreducing the amplification products stained with acridine orange.

EXAMPLE 3 Effect of Another Intercalator On the Detection of the LAMPReaction Product Using Ethidium Bromide

The LAMP reaction was carried out under the same reaction conditions asin Example 1 except that, in addition to ethidium bromide, 20 μM ofmethylene blue, 1 μg/ml of actinomycin D, or 100,000-fold diluted SYBR®Green 2 (Molecular Probes) was added as the second intercalator. Thus,effects of each of the aforementioned second intercalators on theefficiency of detecting the LAMP reaction product were inspected. Theresults are shown in FIG. 5. In the case of the reaction solutions towhich the second intercalator was not added in addition to ethidiumbromide (EtBr in FIG. 5), the fluorescence intensity of the positivereaction solution was 3,085, and that of the negative reaction solutionwas 2,701. That is, difference in the fluorescence intensity resultingfrom the occurrence of products of double-stranded nucleic acidamplification was 384. In contrast, in the case of the reactionsolutions to which methylene blue was added (EtBr+MB in FIG. 5), thefluorescence intensity of the positive reaction solution was 860, andthat of the negative reaction solution was 116. That is, difference inthe fluorescence intensity resulting from the occurrence of products ofdouble-stranded nucleic acid amplification was as Large as 744. In thecase of the reaction solutions to which actinomycin D was added (EtBr+ADin FIG. 5), the fluorescence intensity of the positive reaction solutionwas 3,158, and that of the negative reaction solution was 2,322. Thatis, difference in the fluorescence intensity resulting from theoccurrence of products of double-stranded nucleic acid amplification wasas large as 836. Further, in the case of the reaction solutions to whichSYBR® Green 2 was added (EtBr+SYBR-G in FIG. 5), the fluorescenceintensity of the positive reaction solution was 2,822, and that of thenegative reaction solution was 2,268. That is, difference in thefluorescence intensity resulting from the occurrence of products ofdouble-stranded nucleic acid amplification was as large as 554.

Thus, sensitivity for detecting products of double-stranded nucleic acidamplification using ethidium bromide can be enhanced by performing theLAMP reaction in the presence of the second intercalator together withethidium bromide.

EXAMPLE 4 Effect of Another Intercalator On the Detection of the LAMPReaction Product Using Acridine Orange

The LAMP reaction was carried out under the same reaction conditions asin Example 1 except that, in addition to acridine orange that was addedinstead of ethidium bromide, 20 μM of methylene blue, 1 μg/ml ofactinomycin D, or 100,000-fold diluted SYBR® Green 2 (Molecular Probes)was added as the second intercalator and the assay wavelength was set at575 nm. Thus, effect of each intercalator on the efficiency of detectingthe LAMP reaction product was inspected. The results are shown in FIG.6. In the case of the reaction solutions to which the secondintercalator was not added in addition to acridine orange (AO in FIG.6), the fluorescence intensity of the positive reaction solution was7,121, and that of the negative reaction solution was 6,195. That is,difference in the fluorescence intensity resulting from the occurrenceof products of double-stranded nucleic acid amplification was 926. Incontrast, in the case of the reaction solutions to which methylene bluewas added (AO+MB in FIG. 6), the fluorescence intensity of the positivereaction solution was 3,500, and that of the negative reaction solutionwas 350. That is, difference in the fluorescence intensity resultingfrom the occurrence of products of double-stranded nucleic acidamplification was as large as 3150. In the case of the reactionsolutions to which actinomycin D was added (AO+AD in FIG. 6), thefluorescence intensity of the positive reaction solution was 7,368, andthat of the negative reaction solution was 4,762. That is, difference inthe fluorescence intensity resulting from the occurrence of products ofdouble-stranded nucleic acid amplification was as large as 2,606.Further, in the case of the reaction solutions to which SYBR Green 2 wasadded (AO+SYBR-G in FIG. 6), the fluorescence intensity of the positivereaction solution was 9,435, and that of the negative reaction solutionwas 4,721. That is, difference in the fluorescence intensity resultingfrom the occurrence of products of double-stranded nucleic acidamplification was as large as 4,714.

Thus, sensitivity for detecting products of double-stranded nucleic acidamplification using acridine orange can be enhanced by performing theLAMP reaction in the presence of the second intercalator together withacridine orange.

EXAMPLE 5 Effect of Methylene Blue On the Detection of the LAMP ReactionProduct Using YO-PRO-1®

The LAMP reaction was carried out under the same reaction conditions asin Example 3 except that 1 μg/ml of YO-PRO-1® and 10 μM of methyleneblue as the second intercalators were added instead of ethidium bromide.Thus, the effect of methylene blue on the efficiency of detecting theLAMP reaction product using YO-PRO-1® was inspected. The fluorescenceintensity was assayed using 20 μl of the reaction solution after theamplification and a plate reader (Polarion, Tecan) at an excitationwavelength of 485 nm, at an assay wavelength of 535 nm, and at 25° C.The assay results are shown in FIG. 7.

In the case of the reaction solutions to which methylene blue was notadded (YO-PRO-1® in FIG. 7), the fluorescence intensity of the positivereaction solution was 39,194, and that of the negative reaction solutionwas 34,047. That is, difference in the fluorescence intensity resultingfrom the occurrence of products of double-stranded nucleic acidamplification was 5,147.

In contrast, in the case of the reaction solutions to which methyleneblue was added (YO-PRO-1® +MB in FIG. 7), the fluorescence intensity ofthe positive reaction solution was 33,596, and that of the negativereaction solution was 13,592. That is, difference in the fluorescenceintensity resulting from the occurrence of products of double-strandednucleic acid amplification was as large as 20,004.

Thus, sensitivity for detecting products of double-stranded nucleic acidamplification using YO-PRO-1® can be enhanced by performing the LAMPreaction in the presence of methylene blue as the second intercalatortogether with YO-PRO-1®.

EXAMPLE 6 Effect of a Complex-forming Compound On the Detection of theLAMP Reaction Product Using Methylene Blue

Methylene blue and Acid Orange 7 are mixed in an aqueous solution. Thisforms a complex. Upon the formation of a complex, the absorptionspectrum of methylene blue and that of Acid Orange 7 (AdO) are varied.Thus, the formation of a complex can be confirmed by assaying theabsorption spectrum.

500 μM of methylene blue or a mixed solution of methylene blue and AdO(500 μM each; methylene blue forms a complex with AdO) was added to theLAMP reaction solution, and the resultant was subjected to nucleic acidamplification in the same manner as in Example 1. The absorbance of thereaction solution after the amplification was assayed using a ShimadzuUV-2200 spectrophotometer (using a 10-mm cell) at the assay wavelengthof 680 nm. The assay results are shown in FIG. 8. In the case of thereaction solutions to which AdO was not added (MB in FIG. 8), theabsorbance of the positive reaction solution was 0.75, and that of thenegative reaction solution was 0.71. That is, difference in theabsorbance resulting from the occurrence of products of double-strandednucleic acid amplification was 0.04. In contrast, in the case of thereaction solutions to which AdO was added (MB+AdO in FIG. 8), theabsorbance of the positive reaction solution was 0.46, and that of thenegative reaction solution was 0.38. That is, difference in theabsorbance resulting from the occurrence of products of double-strandednucleic acid amplification was 0.08.

This indicates that AdO regulated the insertion of methylene blue intoDNA. More specifically, the insertion of methylene blue intosingle-stranded DNA is weak and thus is inhibited by AdO. On thecontrary, the insertion of methylene blue into double-stranded DNA isstrong and thus cannot be inhibited by AdO. Thus, methylene blue isconsidered to be released from a complex.

Therefore, a bond of an intercalator to a single-stranded nucleic acidis preferentially inhibited by the formation of a complex as well as byoxidation or reduction. Thus, the sensitivity of detecting products ofdouble-stranded nucleic acid amplification can be enhanced.

All publications, patents, and patent applications cited herein areincorporated herein by reference in their entirety.

INDUSTRIAL APPLICABILITY

The present invention provides a method for efficiently detectingproducts of double-stranded nucleic acid amplification. In particular,the application of the present invention to the detection of products ofnucleic acid amplification by the LAMP method can effectively reducebackground noises, which are derived from single-stranded nucleic acidcaused by the use of a larger amount of primers compared with the caseof PCR.

Free Text of Sequence Listing

SEQ ID NO: 1; synthetic DNA

SEQ ID NO: 2; synthetic DNA

SEQ ID NO: 3; synthetic DNA

SEQ ID NO: 4; synthetic DNA

SEQ ID NO: 5; synthetic DNA

1. A method for detecting a product of nucleic acid amplificationcomprising the following steps: (a) amplifying a nucleic acid throughnucleic acid amplification; (b) adding an intercalator to a reactionsolution after the nucleic acid amplification; (c) reducing signalsderived from an intercalator bound to a single-stranded nucleic acid byadding a compound that reacts preferentially with an intercalator boundto a single-stranded nucleic acid as compared to an intercalator boundto a double-stranded nucleic acid to a mixture comprisingdouble-stranded nucleic acid bound to an intercalator andsingle-stranded nucleic acid bound to an intercalator, thereby reducingsignals derived from an intercalator bound to a single-stranded nucleicacid; and (d) assaying the fluorescence intensity of a reactionsolution.
 2. A method for detecting a product of nucleic acidamplification, the method comprising: (a) amplifying a nucleic acidthrough nucleic acid amplification in the presence of an intercalator;(b) reducing signals derived from an intercalator bound to asingle-stranded nucleic acid by adding a compound that reactspreferentially with an intercalator bound to a single-stranded nucleicacid as compared to an intercalator bound to a double-stranded nucleicacid to a mixture comprising double-stranded nucleic acid bound to anintercalator and single-stranded nucleic acid bound to an intercalator,thereby reducing signals derived from an intercalator bound to asingle-stranded nucleic acid; and (c) assaying the fluorescenceintensity of a reaction solution.
 3. The method for detection accordingto claim 1, wherein the nucleic acid amplification is carried out by thefollowing steps: (a) selecting a first arbitrary sequence F1c, a secondarbitrary sequence F2c, and a third arbitrary sequence F3c in that orderfrom the 3′ terminus in a target region toward the 3′ terminus on thepolynucleotide chain and a fourth arbitrary sequence R1, a fiftharbitrary sequence R2, and a sixth arbitrary sequence R3 in that orderfrom the 5′ terminus in the target region toward the 5′ terminus of thenucleotide chain; (b) preparing a primer containing sequence F2 which iscomplementary to F2c and, on the 5′ side of F2, the same sequence asF1c; a primer containing sequence F3 which is complementary to F3c; aprimer containing the same sequence as R2 and, on the 5′ side of thesequence, sequence R1c which is complementary to R1; and a primercontaining the same sequence as R3; and (c) synthesizing DNA in thepresence of a strand displacement-type polymerase and the primers usingthe nucleotide chain as a template.
 4. The method for detectionaccording to claim 1, wherein the nucleic acid amplification is carriedout by the following steps: (a) selecting a first arbitrary sequence F1cand a second arbitrary sequence F2c in that order from the 3′ terminusin a target region toward the 3′ terminus on the polynucleotide chainand a third arbitrary sequence R1 and a fourth arbitrary sequence R2 inthat order from the 5′ terminus in the target region toward the 5′terminus of the nucleotide chain; (b) preparing a primer containingsequence F2 which is complementary to F2c and, on the 5′ side of F2, thesame sequence as F1c; and a primer containing the same sequence as R2and, on the 5′ side of the sequence, sequence R1c which is complementaryto R1; and (c) synthesizing DNA in the presence of a stranddisplacement-type polymerase, the primers, and a melting temperatureregulator using the nucleotide chain as a template for amplification. 5.The method for detection according to claim 4, wherein the meltingtemperature regulator is betaine or trimethylamine N-oxide.
 6. Themethod of claim 1, wherein the intercalator is any of ethidium bromide,acridine orange, TO-PRO-1® (Quinolinium, 4-[(3-methyl-2(3H)-benzothiazolylidene)methyl]-1-[3-(trimethylammonio)propyl]-, diiodideYO-PRO-1® (Quinolinium,4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1-[3-(trimethylammonio)propyl]-,diiodide), or methylene blue.
 7. The method of claim 1, wherein thecompound that reacts preferentially with an intercalator bound to asingle-stranded nucleic acid as compared to an intercalator bound to adouble-stranded nucleic acid is an oxidant or reducer.
 8. The method ofclaim 7, wherein the oxidant is any of sodium hypochlorite, hydrogenperoxide, or potassium permanganate.
 9. The method of claim 7, whereinthe reducer is sodium borohydride or sodium cyanoborohydride.
 10. Themethod of claim 2, wherein the intercalator is any of ethidium bromide,acridine orange, TO-PRO-1® (Quinolinium, 4-[(3-methyl-2(3H)-benzothiazolylidene)methyl]-1-[3-(trimethylammonio)propyl]-, diiodide),YO-PRO-1® (Quinolinium,4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1-[3-(trimethylammonio)propyl]-,diiodide), or methylene blue.
 11. The method of claim 2, wherein thecompound that reacts preferentially with an intercalator bound to asingle-stranded nucleic acid as compared to an intercalator bound to adouble-stranded nucleic acid is an oxidant or a reducer.
 12. The methodof claim 11, wherein the oxidant is any of sodium hyperchlorite,hydrogen peroxide, or potassium permanganate.
 13. The method of claim11, wherein the reducer is sodium borohydride or sodiumcyanoborohydride.
 14. The method of claim 2, wherein the compound thatis bound to a single-stranded nucleic acid more strongly than anintercalator and is bound to a double-stranded nucleic acid more weaklythan an intercalator is a second intercalator different from saidintercalator.
 15. The method of claim 14, wherein the secondintercalator is any of methylene blue, actinomycin D, SYBR® Green 2 (CASRegistry No. 172827-25-7) or OliGreen® (CAS Registry No. 268220-33-3).